In plants, ethylene (C2H4) is a regulator of various physiological and morphological responses, including inhibition of cell expansion, promotion of leaf and flower senescence, induction of fruit ripening and abscission, resistance to pathogen infection, and adaptation to stress conditions (Bleecker, A. B. and Kende, H., Annu. Rev. Cell. Dev. Biol., 16:1-18 (2000); Guo, H. and Ecker, J. R., Curr. Opin. Plant. Biol., 7:40-49 (2004)). The molecular dissection of ethylene signal transduction began with genetic screens based on the well-documented triple response phenotype of ethylene-treated etiolated Arabidopsis seedlings. Through these screens, many ethylene mutants have been obtained, including the ethylene insensitive mutants etr1, ein2, ein3, ein5 (Bleecker, A. B. et al., Science, 241:1086-1089 (1988); Guzman, P. and Ecker, J. R., Plant Cell, 2:513-523 (1990); Roman, G. et al., Genetics, 139:1393-1409 (1995)); the ethylene overproducing mutants eto1, eto2, eto3, and the ethylene constitutive response mutant ctr1 (Guzman, P. and Ecker, J. R., Plant Cell, 2:513-523 (1990); Kieber, J. J. et al., Cell, 72:427-441 (1993)). Initial studies of these mutants have revealed a mostly linear framework for the ethylene-signaling pathway, leading from ethylene perception at the membrane to transcriptional activation in the nucleus (Stepanova, A. N. and Ecker, J. R., Curr. Opin. Plant. Biol., 3:353-360 (2000); Chen, Y. F. et al., J. Biol. Chem., 277:19861-19866 (2002); Guo, H. and Ecker, J. R., Curr. Opin. Plant. Biol., 7:40-49 (2004)).
Ethylene is perceived by a family of membrane bound, endoplasmic reticulum-located receptors ETHYLENE RESPONSE1 (ETR1), ETHYLENE RESPONSE SENSOR1 (ERS1), ETHYLENE RESPONSE2 (ETR2), ETHYLENE INSENSITIVE4 (EIN4), and ETHYLENE RESPONSE SENSOR2 (ERS2), which are similar in sequence and structure to bacterial two-component histidine kinases (Chang, C. et al., Science, 262:539-544 (1993); Hua, J. et al., Plant Cell, 10:1321-1332 (1998); Kendrick, M. D. and Chang, C., Curr. Opin. Plant. Biol., 11:479-485 (2008)). Each receptor has an N-terminal membrane-spanning domain that binds ethylene with a copper cofactor provided by the RESPONSIVE TO ANTAGONIST1 (RAN1) copper transporter (Hirayama, T. et al., Cell, 97:383-393 (1999)). Briefly, in the absence of ethylene gas, the ethylene receptors repress downstream responses through interaction with CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) (Gao, Z. et al., J. Biol. Chem., 278:34725-34732 (2003)), which is a member of Raf kinase family that also acts as a negative regulator of the downstream ethylene signaling pathway (Kieber, J. J. et al., Cell, 72:427-441 (1993)). In the presence of ethylene, the receptors stop repressing ethylene response through inactivation of CTR1. Additionally, EIN2 is de-repressed and positively regulates the levels of ETHYLENE INSENSITIVE3 (EIN3) and ETHYLENE INSENSITIVE3-LIKE1 (EIL1) the key transcription factors of ethylene signaling pathway, which results in the activation of transcription of ethylene responsive genes (Chao, Q. et al., Cell, 89:1133-1144 (1997); Solano, R. et al., Genes & Dev., 12:3703-3714 (1998)). Recently, numerous studies have expanded the linear view of ethylene signaling pathway. For instance, a new protein, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), which is co-localized with the ethylene receptor ETR1, was identified as a positive regulator of ETR1 function, but the connection between RTE1 and ETR1 is still under investigation (Resnick, J. S. et al., Proc. Natl. Acad. Sci. U.S.A., 103:7917-7922 (2006); Solano, R. et al., Genes & Dev., 12:3703-3714 (1998); Dong, C. H. et al., Plant J., 53:275-286 (2008)). Additionally, a number of groups found that posttranscriptional regulation of protein levels is a key mechanism of modulating EIN3 activity by ethylene. Specifically, they found that ubiquitin/proteasome-mediated degradation negatively regulates ethylene responses by targeting EIN3 for turnover through two F-box proteins EIN3-BINDING F BOX PROTEIN1 (EBF1) and EIN3-BINDING F BOX PROTEIN2 (EBF2) (Guo, H. and Ecker, J. R., Cell, 115:667-677 (2003); Potuschak, T. et al., Cell, 115:679-689 (2003); Gagne, J. M. et al., Proc. Natl. Acad. Sci. U.S.A., 101:6803-6808 (2004)). Interestingly, negative feedback regulation exists in this step of the ethylene signal-transduction pathway, in that EIN3 targets the promoter of EBF2 to control its expression level likely allowing fine-tuning of ethylene responses (Binder, B. M. et al., Plant Cell, 19:509-523 (2007); Konishi, M. and Yanagisawa, S., Plant J., 55:821-831 (2008)). Most recently, an alternative ethylene signaling pathway has been proposed that is based on studies of ethylene responses in Arabidopsis protoplasts (Varma Penmetsa, R. et al., Plant J., 55:580-595 (2008); Yoo, S. D. et al., Nature, 451:789-795 (2008)). Characterization of these genes/proteins has provided additional insight into the molecular mechanisms that may underlie the response of plants to ethylene gas.
EIN2 is an integral membrane protein with limited similarity in the N-terminus to mammalian NRAMP metal transporters, the <850 amino acid C-terminus of EIN2 is conserved in all the known EIN2 homologs of angiosperms (Varma Penmetsa, R. et al., Plant J., 55:580-595 (2008)). Interestingly, expression of a portion of the C-terminus (EIN2-CEND) is sufficient to constitutively activate ethylene and stress responses both in Arabidopsis (Alonso, J. M. et al., Science, 284:2148-2152 (1999)) and in Medicago (Mt) (Varma Penmetsa, R. et al., Plant J., 55:580-595 (2008)). Phenotypic, epistatic and biochemical analyses place EIN2 in a central position in ethylene signaling pathway (Roman, G. et al., Genetics, 139:1393-1409 (1995); Johnson, P. R. and Ecker, J. R., Annu. Rev. Genet., 32:227-254 (1998); Guo, H. and Ecker, J. R., Cell, 115:667-677 (2003)).